Spermatogenesis has been known for decades to depend on androgens, but the molecular mechanism behind this has remained largely elusive. Even the cell types in the testis that drive spermatogenesis in response to androgens had not been clearly defined until recent mouse genetic studies demonstrated that at least one participant is the Sertoli cell. It is logical that the Sertoli cell would promote spermatogenesis in response to androgens, as it is in intimate contact with developing germ cells and expresses high levels of androgen receptor (AR), a nuclear hormone-receptor family member and transcription factor that must usually be bound to androgen to activate its target genes. Most androgen-regulated genes in Sertoli cells may not be direct targets of AR but instead may be regulated by androgen-regulated transcription factors. These secondary androgen-response genes have cis elements that bind to androgen-regulated transcription factors. To date, no transcription factors regulating these secondary androgen-response genes in Sertoli cells have been definitively identified. A good candidate is the founding member of the Rhox homeobox gene cluster, Rhox5 (Pem), as it is androgen regulated, selectively expressed in Sertoli cells, and necessary for normal spermatogenesis. This proposal focuses on two other androgen-regulated genes in the Rhox homeobox gene cluster, Rhox10 and Rhox11. Each is expressed at peak levels in the testes at postnatal time points that correspond to two distinct AR-dependent events during spermatogenesis: the progression of male germ cells through meiosis I and the transition of late-round spermatids into elongating spermatids. Thus, we hypothesize that Rhox10 and Rhox11 encode transcription factors that participate in mediating these two AR-dependent events. The Specific Aims of this application are (i) to identify the functions of the androgen-regulated Rhox10 and Rhox11 genes in spermatogenesis and sperm maturation in vivo;(ii) to distinguish the independent and redundant roles of the androgen-regulated Rhox5, Rhox10, and Rhox11 genes in male reproduction;and (iii) to begin to elucidate the AR-dependent gene networks that drive spermatogenesis by identifying gene targets of the androgen-regulated Rhox genes. By providing a molecular basis for androgen-driven spermatogenesis, the proposed work could ultimately lead to cures for some cases of male infertility and the development of novel male contraceptive methods.